Humoral immunogenicity assessment after receiving three types of SARS-CoV-2 vaccine

Several vaccines have been developed against SARS-CoV-2 and subsequently approved by national/international regulators. Detecting specific antibodies after vaccination enables us to evaluate the vaccine’s effectiveness. We conducted a prospective longitudinal study among members of Tarbiat Modares University of Tehran, Iran, from 4 September 2021 until 29 December 2021. We aimed to compare the humoral immunogenicity of 3 vaccine types. Participants consisted of 462 adults. Anti-SARS-CoV-2 receptor-binding domain [RBD] IgG titer was compared in 3 groups, each vaccinated by available vaccines in Iran at the time: Oxford/AstraZeneca, COVIran Barekat, and Sinopharm. The median IgG titer was: 91.2, 105.6, 224.0 BAU/ml for Sinopharm, COVIran Barekat and Oxford/AstraZeneca respectively after the first dose; 195.2, 192.0, 337.6 BAU/ml after the second one. We also analyzed the frequency of antibody presence in each vaccine group, in the same order the results were 59.0%, 62.6% and 89.4% after the first dose and 92.1%,89.5% and 98.9% after the second. The comparison of results demonstrated that AstraZeneca vaccine is a superior candidate vaccine for COVID-19 vaccination out of the three. Our data also demonstrated statistically significant higher antibody titer among recipients with an infection history.


Demographic characteristics of participants
A total of 462 participants were included in this study, who received 3 different types of approved and available SARS-CoV-2 vaccines in Iran.The participant's ages ranged from 20 to 70 years old with a median of 37 (IQR 30-45), 44.2% of them were female, and 10.6% of all of the participants had priorSARS-CoV-2 infection history.In terms of age, sex, or infection history distribution, there were no statistically significant differences observed among the participants in the 3 vaccine groups.The demographic characteristics of participants are demonstrated in Table 1.Out of 462 participants, 301 returned for secondary examinations.Their demographic information is demonstrated in Table 2.

Humoral immunogenicity assessment
Serum antibody titers were measured one month after each of the first and second vaccine inoculation.The median of antibody titers and IQR are demonstrated in Table 3.Because the non-normal distribution of antibody titers the non-parametric analysis, Kruskal-Wallis, was used to compare antibody titers among the 3 vaccine Table 1.Demographic characteristics of the participants receiving the first dose of COV2-vaccination (n = 462).SD, standard deviation.www.nature.com/scientificreports/types, which showed P-values < 0.001 after both the first and the second dose of vaccination.Since this study includes three vaccine types, in order to do pairwise comparisons and determine the significant differences in their effectiveness, the Bonferroni Correction test was applied.This test showed that the difference in antibody titers in the COVIran Barekat-Sinopharm pair had an adjusted P-value of 1.000 which is not significant, while Sinopharm-AstraZeneca and COVIran Barekat-AstraZeneca pairs both had P-values < 0.001.As was expected, since Sinopharm and COVIran Barekat both use a similar production strategy, killed whole virion vaccine, induced similar antibody titers.The frequency and percentage of participants who tested positive for anti-SARS-CoV-2 anti-S1-RBD antibody in each vaccine group are shown in Table 4. Chi-Square test was utilized to survey for any significant differences between the three different vaccine groups.The results demonstrated a statistically significant difference between positive results among participants in the 3 vaccine groups in both doses of vaccines, P-value < 0.001 for the first and P-value: 0.023 for the second dose were calculated.

Impact of infection history, age and sex
We also assessed the impact of infection history on antibody titer through the Mann-Whitney U test for each vaccine group.After the first vaccination, distribution of antibody titer was significantly different based on infection history in AstraZeneca, Sinopharm, and the overall groups.Antibody titers were higher in participants with prior covid-19 infection.On the contrary, it was the same across categories of infection history for COVIran Barekat.After the second dose, compared to non-infected participants, our data demonstrated significant differences in antibody titers among recipients with an infection history, only in AstraZeneca and overall groups

Discussion
Humoral immunity is a major factor which plays a critical role in vaccines effectiveness.Despite the fact that the exact neutralizing response is investigated thorough cVNT, which requires biosafety level 3 equipment, Anti-SARS-CoV-2 anti RBD titer can be measured to evaluate humoral responses after infection or vaccination.The sVNT is practical in numerous studies on features of COVID-19, including vaccine efficacy during preclinical and clinical trials of different vaccine candidates and monitoring neutralizing antibody titers in vaccine recipients after mass vaccination 7,22,23 .This was a prospective longitudinal study performed on 462 adult participants each receiving 2 doses of either AstraZeneca, COVIran Barekat or Sinopharm vaccines.One month after the injection offirst dose, a significant statistical difference was shown when comparing inactivated vaccine recipients (COVIran Barekat or Sinopharm) with participants who received AstraZeneca vaccine, both in the antibody presence (AstraZeneca 89.4%, COVIran Barekat 62.6%, and Sinopharm 59.0%, P-Value < 0.001) and the median antibody titer (AstraZeneca 224.0 (IQR 68.8-396.8)BAU/ml, COVIran Barekat 105.6 (IQR 16.0-284.8)BAU/ml, and Sinopharm 91.2 (IQR 16.0-276.8)BAU/ml, P-Value < 0.001).Also, the same results were obtained after the injection of the second dose, antibody presence were as follows: AstraZeneca 98.9%, COVIran Barekat 89.5%, and Sinopharm 92.1% (P-Value < 0.001).Also, the median antibody titer was significantly higher in AstraZeneca recipients (AstraZeneca 377.6 (IQR 275.2-416.0)BAU/ml, COVIran Barekat 192.0 (IQR 72.0-297.6)BAU/ml, and Sinopharm 195.2 (IQR73.6-302.4)BAU/ml, P-Value < 0.001).Inevitably, the protection offered by neutralizing antibodies through vaccination can be varied due to the differences in vaccine platforms and the interval of doses.Our findings demonstrated that Astra Zeneca has led to higher immunogenicity compared to other available vaccines in Iran.
An Iranian cohort monitoring study has revealed that when comparing AstraZeneca recipients with inactivated vaccines recipients, although the former has a significantly higher immunity during the interval of the first and second dose, after the second dosage no significant difference was found among different vaccine platforms.They concluded that SARS-CoV-2 vaccines, while unable to prevent the infection, can significantly reduce mortality rate 24 .These results are in line with our findings about the higher anti-SARS-CoV-2 anti RBD IgG detection rate and titer in AstraZeneca recipients.
It is obvious that antibody titers, post-infection or vaccination, cause a range of protective effects specifically by reducing the mortality rate.A study estimated that the required neutralizing antibody titer to completely prevent symptomatic viral infection is approximately sixfold higher compared to what is required to prevent severe outcomes of SARS-CoV-2 infection 25 .
In this study, we assessed antibodies in participants infected or not infected early in the pandemic during alfa and delta variants circulation time.The assessment of serum antibody activity after the first dose of SARS-CoV-2 vaccination (in September 2020) showed that participants who had previously confirmed infection in each vaccine group, or overall had a significantly higher amount of antibody titer as demonstrated in the RBD-based ELISA assay, except for COVIran Barekat group, which might be due to the low number of infected persons in this group: 11 out of 155 (7.09%).Moreover 2 of 11 COVIran Barekat recipients who had prior infection history, did not produce antibodies after the first vaccine administration.Considering that both of them were young (a 30 years old female and 38 years old male), they might be non-responders.The impact of these two samples was magnified in such a small fraction.Therefore, when we used Whitney U test for each vaccine group in order to investigate the impact of infection history, it led to a non-significant p-value for COVIran barekat group.These findings confirmed that additional antigenic exposure further improves antibody efficacy against SARS-CoV-2 variants.Protective antibodies from prior infection, with or without vaccination, induced antibodies against the spike protein to interfere with its function 26 .The antibody response is a crucial aspect of adaptive immunity against viral infections.Based on the predominant isotypes and profiles of somatic hypermutations of the antibodies, the humoral immune response can be divided into two phases.In the extrafollicular (EF) phase, B cells are activated and quickly differentiate into plasma cells in foci outside of the follicle within a few days after the infection.They produce antibodies that contain few somatic hypermutations but can still have reasonably high affinities and neutralize the virus 27 .Another study revealed the chances of reinfection are significantly lower in individuals who tested positive for antibodies.Furthermore, getting vaccinated after a SARS-CoV-2 infection enhances the impact of this response.Research has found that neutralizing antibodies are present in 99% of www.nature.com/scientificreports/people infected with SARS-CoV-2 before.It is believed that the antibodies could provide strong protection against infection and reinfection in individuals without COVID-19 infection history, but were vaccinated previously 28 .Many scientists are of the same concern about the waning of neutralizing antibody titers induced through prior infection or vaccination.Besides, the continuous emerge of new Variants Of Concern (VOC) of SARS-CoV-2 due to its broad spread and frequent mutation, has caused several neutralizing antibodies to lose their antiviral activities.However, neutralizing antibodies can recognize different epitopes in spike protein and some of them can neutralize new VOCs.As a result of that, pervious immunity induced by infection or vaccine might alleviate infection.It was suggested that, in order to decrease the VOC's chance of escape from vaccine induced neutralizing antibodies, future vaccines must consider different groups of epitopes of RBD 29 .
The next step is to develop vaccines which are able to prevent infection completely.Mucosal immunity inducing vaccines might be effective in preventing infection and restricting viral transmission, however, there are still a number of obstacles to overcome to reach their full potential 30 .Mucosal vaccines have been successful in protecting against diseases, but there are still limitations and risks associated with their clinical development.One concern is that mucosal surfaces are frequently exposed to harmless foreign substances and commensals, which can lead to tolerogenic immune programs that may result in weak induction of pro-inflammatory responses.Effective adjuvants can help break tolerance, however, there are limited mucosal adjuvants that are safe for human use and many require further testing to demonstrate their safety and efficacy.Additionally, antigen dilution can hinder vaccine delivery at mucosal surfaces, where ciliated cells quickly clear away debris and pathogens in a protective mucous layer 31 .
In this study, previous infection history was significantly related to higher anti SARS-CoV-2 anti RBD IgG titers (P-Value < 0.001 after the first dose, P-Value: 0.002 after the second dose).Many studies subscribe to this theory, which states infection history can boost vaccine effectiveness and lead to an increase in antibody titer and furthermore can shorten the interval of vaccine injection and rises in antibody titer 21,32,33 .
After categorizing participants based on their sex or age, no significant difference was observed in the median antibody titer.AstraZeneca clinical trial phase 2/3 data has not shown any significant difference in neutralizing antibody titer among different age groups, after the second dose injection.The data also demonstrated that neutralizing antibody has been detected among 99% of participants 14 days after the second dose injection 34 .However, another meta-analysis including approximately 7 million participants from 18 studies, has found that vaccine effectiveness in prevention of severe SARS-CoV-2 infection was significantly lower among participants older than 65 years old 35 .It might be due to the fact that different vaccine types were investigated in that study; since it investigated mRNA-based vaccines and Johnson and Johnson vaccine.Moreover, our study's aim was to evaluate antibody titer among university staff and students, leading to 84% of our participants being 50 years old or younger, which may result in limitations.
The limitations of this study are: it did not consider children and elderly citizens (> 70), cell-mediated immune responses were not assessed, and last but not least, 34% of the participants in the first sampling did not return to partake in the second round of it.
Although, the emergence of new VOCs and waning neutralizing activity of vaccines has faced researchers with a dilemma, how often should we administer SARS-CoV-2 vaccines and more importantly how safe are these repeated injections?Further studies need to address the problems of safety and efficacy of the developed and the under-development vaccines against SARS-CoV-2, which are achievable through real-world surveys.

Conclusion
This study demonstrated that AstraZeneca vaccine induced higher humoral immunogenicity when compared to the inactivated vaccines (COVIran Barekat, Sinopharm), since it had more positive cases of antibody presence and higher antibody.Also, results of this study, agreeing with several other studies, indicated that infectioninduced immunity combined with vaccination results in higher levels of protection against COVID-19 which may prevent severe illness, hospitalization and mortality.Therefore, our findings suggested that vaccination may be more crucial for people who haven't been infected by SARS-CoV2.

Study design
This research was a prospective longitudinal study, performed among 462 staff and students of Tarbiat Modares University, Tehran, Iran.It was conducted from 4 September 2021 until 29 December 2021.The Delta variant of SARS-CoV-2 was the predominant strain during this period.
All university staff and students who attended the health center of Tarbiat Modares University to get their second dose of vaccination with Oxford/AstraZeneca, COVIran Barekat or Sinopharm, and were willing to participate in the study were enrolled.2 ml of peripheral venous blood sample was collected for serological survey.The participants were recalled 1 month after the second injection for the second round of blood sample collection.

Ethical considerations
This study was performed in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Tarbiat Modares University of Tehran (code number: IR.MODARES.REC.1400.138).
The researchers explained the method and aim of the study.The participants signed the formal informed consent form and were able to withdraw from the study at any time.They were also able to communicate with the research team for more information about the tests and results, or ask any questions related to this study. https://doi.org/10.1038/s41598-023-47611-w

Table 2 .
Demographic characteristics of the participants receiving the second dose of COV2-vaccination (n = 301).SD, standard deviation.

Table 3 .
Comparison of antibody titers after the first and second dose of COV2-vaccinations.IQR, interquartile range; a ; antibody titer (BAU/ml) before the second dose of vaccination; b ; antibody titer (BAU/ ml) 1 month after the second dose of vaccination.Significance values have been adjusted by the Bonferroni correction for multiple tests (Sinopharmm-COVIran Barekat 1.000, Sinopharm-AstraZeneca < 0.001, COVIran Barekat-AstraZeneca < 0.001) for both of measurements.

Table 4 .
Comparison of antibody presence in different COV2-vaccines.

Table 5 )
. Spearman's method was used to assess the correlation between antibody titer and age for each vaccine group.Besides, we evaluated the influence of sex on antibody titer by Mann Whitney U test for each vaccine group.Both analyses failed to demonstrate statistically significant relationships.

Table 5 .
The effect of infection history on antibody titer in 3 vaccine types.Inf.h, infection history.